Cytotoxic activity of the genus Ferula (Apiaceae) and its bioactive constituents.

Objective
The genus Ferula L. includes perennial flowering plants belonging to the Apiaceae family. This genus is a rich source of biologically active phytochemicals such as sulfur-containing derivatives, coumarins, sesquiterpenes, sesquiterpene lactones, sesquiterpene coumarins, glucuronic acid, galactose, arabinose, rhamnose, and daucane esters. Over the last decade, considerable attention has been paid to biological activities of these compounds; it is assumed that the most prominent biological features of the genus Ferula are their cytotoxic effects. This article discusses cytotoxic activity of the genus Ferula and their important compounds.


Materials and Methods
In this mini-review article, papers published from 1990 to April 2016 were included and the following information was discussed; cytotoxic activity of the genus Ferula and their important compounds, the type of cell line used in vitro, concentrations of the extracts/active compound that were used, and the underlying mechanisms of action through which Ferula-related chemicals induced cytotoxicity. In addition, we explained different mechanisms of action through which the active constituents isolated from Ferula, could decrease cellular growth.


Conclusion
It is highly recommended that potent and effective compounds that were isolated from Ferula plants and found to be appropriate as adjuvant therapy for certain diseases, should be identified. Also, the versatile biological activities of sesquiterpene coumarins suggest them as promising agents with a broad range of biological applications to be used in the future.


Introduction
The genus Ferula includes perennial flowering plants belonging to the family Apiaceae (Umbelliferae). This genus consists of about170 species which are distributed worldwide. Out of 30 species of Ferula that could be found in Iran, 16 plants are endemic. Different species of the genus Ferula are broadly distributed in arid areas from the eastern Mediterranean regions to central Asia (Gholami and Shamsara, 2016;Karimi et al., 2010;Nazari and Iranshahi, 2011); however, some Ferula species are found in arid regions of temperate Eurasia, in the Canary Islands and in North Africa (e.g. Tunisia) (Znati et al., 2014). Different species of the genus Ferula are regarded as rich sources of biologically active phytochemicals such as sulfur-containing derivatives, coumarins, coumarin esters, sesquiterpenes, sesquiterpene lactones, sesquiterpene coumarins, glucuronic acid, galactose, arabinose, rhamnose, and daucane esters ( Figure. 1) (Asghari et al., 2016;Maggi et al., 2016;Nazari and Iranshahi, 2011;Razavi et al., 2016).
Some species of the genus Ferula have therapeutic properties such as contraceptive, antipyretic, smooth-muscles relaxant and aphrodisiac activities (Nazari and Iranshahi, 2011;Yaqoob et al., 2016). Also, several Ferula species are wellknown because of their applications in the treatment of various diseases. For example, F. persica root extract possesses antispasmodic, carminative, laxative and expectorant properties and has been used for the treatment of diabetes and high blood pressure (Razavi and Janani, 2015). F. assa-foetida exhibits anti-carcinogenic properties and has protective activities against free radical-mediated diseases (Gamal-Eldeen and Hegazy, 2010). Iranshahi et al. reported that F. assa-foetida has anti-leishmanial activity against promastigotes (Iranshahi et al., 2007). Moreover, Ferula species have been used in traditional medicine for the treatment of skin infections, hysteria and stomach disorders. Also, a number of Ferula species has been utilized as febrifuge and carminative agents and for relaxation of tracheal smooth muscles (Gamal-Eldeen and Hegazy, 2010). F. assa-foetida and F. gummosa are two famous species of Ferula in Iranian folk medicine. Additionally, some Ferula species are well-known as important sources of aromatic resins and are employed in cosmetic industries (Kanani et al., 2011).
Phytochemicals obtained from the species of Ferula are used in traditional medicine for the treatment of various diseases such as digestive disorders, rheumatism, headache, neurological disorders, arthritis, dizziness and dysentery. Galbanum, the aromatic gum resin obtained from F. gummosa, has been traditionally used as a tonic, anticonvulsant, and emmenagogue agent . Moreover, as asafoetida as the dried latex (gum oleoresin) exudates from the rhizome or tap root of F. assa-foetida, has been traditionally used for the treatment of various diseases including asthma and gastrointestinal disorders as well as removal of intestinal parasites. Asafoetida has also been known to possess antifungal, anti-diabetic, anti-inflammatory, antimutagenic and antiviral activities (Iranshahy and Iranshahi, 2011;Mahendra and Bisht, 2012).
A number of sesquiterpenes obtained from the species of Ferula roots, revealed antibacterial, antifungal, cytotoxic, antioxidant, and hormonal activities as well as P-glycoprotein inhibitory and immunomodulatory effects (Miski, 2013). Sanandajin and ethyl galbanate, the two sesquiterpene coumarins isolated from F. pseudalliacea root extract have shown potent antibacterial activities and are being used in pharmaceutical and food industries (Dastan et al., 2016).
In this review, we focused on cytotoxic activity of Ferula plants reported from 1990 to April 2016.

Group D-Monoterpenoids
Group E-Other compounds Figure1. Chemical structure of some constituents of Ferula categorized in groups A-E.
Feselol and mogoltacin are two biologically active sesquiterpene coumarins isolated from root extracts of Ferula species that showed cytotoxic properties. For example, a combination of 40 mg/mL vincristine and 16 mg/mL mogoltacin increased the cytotoxicity of vincristine by 32.8%, in human transitional cell carcinoma (TCC) cells (BehnamRassouli et al., 2009). Similar results were found for feselol, a sesquiterpene coumarin isolated from the fruits of F. badrakema (Mollazadeh et al., 2010). Also, a combination of feselol and mogoltacin enhanced the cytotoxicity of cisplatin in 5637 cells (human bladder carcinoma cell line) Rassouliet al., 2011). Hanafi-Bojd et al. (2011) showed that farnesiferol A and galbanic acid, two sesquiterpene coumarins isolated from Ferula species, increase verapamil cytotoxicity (Hanafi-Bojd et al., 2011). In another study, sanandajin, farnesiferol B, and kamolonol acetate displayed cytotoxic activities against HeLa cells with IC50 values of 2.2, 6.7, and 4.9 μM, respectively (Dastan et al., 2014). Kasaian et al. (2015) revealed that sesquiterpene coumarins isolated from Ferula species exert different cytotoxic activities. Also, they reported that farnesiferol B, farnesiferol C and lehmferin reverse doxorubicin-resistance properties of MCF-7/Adr cells .
They revealed that these compounds have no significant cytotoxic activities (IC50 values>100 mM) against MCF-7 and K562 cancer cells (Chitsazian-Yazdi et al., 2015). Ferutinin is a natural product isolated from F. ovina possesses apoptosis-inducing effects.
Also, ferutinin analogues synthesized by esterification of jaeschkenadiol using different acids, have exhibited potent inhibitory activity against MCF-7 with an IC50 value of 1 μm Safi et al., 2015).
A number of sesquiterpene lactones isolated from F. oopoda showed significant cytotoxicity. For example, dehydrooopodin revealed significant cytotoxicity with IC50 values of 5 and 15 µM against K562 and MCF7cancer cell lines, respectively (Kasaian et al., 2014).
Cytotoxicity of some sesquiterpene coumarins isolated from F. sinkiangensis was investigated by Li et al., 2015. They found that these sesquiterpene coumarins had selective cytotoxic activity against HeLa and AGS cancer cell lines, with IC50 values of 12.7-226.6 μM (Li et al., 2015).
In 2006, it was reported that compounds isolated from F. assa-foetida have potent and specific NF-κB-inhibiting properties, but their cytotoxicity were negligible (Appendino et al., 2006). Chimgin and chimganin, two monoterpenoid compounds isolated from F. szowitsiana, showed cytotoxic activities. Chimgin showed IC50 values of 45.2, 67.1 and 69.7 µM and chimganin showed IC50values of 28, 74 and 30.9 μM for MCF-7, HepG2 and MDBK cancer cell lines, respectively. These values were just slightly lower than those of tamoxifen which was used as positive control (Sahranavard et al., 2009).
In a number of investigations, Ferula root extracts and fractions have been studied. Eslami et al. (2015) showed that F. gummosa extract has specific cytotoxic effects mainly against MCF7 and oral cancer cell lines (Eslami et al., 2013;Gudarzi et al., 2015). Elouzi et al. (2008) proved that petroleum extract of F. hermonisat the concentration of 0.125 mg/ml, causes 50% cell death (Elouzi et al., 2008).
The extract of F. szowitsiana root was shown to be active against three cancerous (MCF7, HepG2 and WEHI164) and one normal (MDBK) cell lines. In another study, the cytotoxicity of some of the Iranian medicinal Ferula species was examined and all the extracts and oleo-gum resins of F. assa-foetida showed dosedependent cytotoxicity (Bagheri et al., 2010). Hajimehdipoor et al. (2012) investigated the cytotoxic effects of F. persica and F. hezarlalezarica, two endemic Ferula species of Iran, against MCF7, HepG2, HT29 and A549 (adenocarcinomic human alveolar basal epithelial cells), cancer cell lines. They revealed that hexane and chloroform fractions of these plants have cytotoxic effects at concentration up to 100 μg/ml. They also reported that the cytotoxicity of F. persica extracts was higher than that of F. hezarlalezarica extracts (IC50: 22.3-71.8 μg/ml for F. persica and 76.7-105.3 μg/ml for F. hezarlalezarica) (Hajimehdipoor et al., 2012).
In an investigation, F. assa-foetida extract displayed neuroprotective effects in a glutamate-induced neurotoxicity model (Tayeboon et al. 2013). In another study, researchers reported the cytotoxic activities of the extracts and fractions of F. szowitsiana, F. hirtella and F. oopoda against MCF-7, HT-29, A549 and HepG2 cancer cell lines. Based on their data, nhexane and chloroform fractions of F. szowitsiana and F. hirtella were cytotoxic, probably due to the presence of nonpolar/semi-polar constituents (Hamzeloomoghadam et al. 2013). Furthermore, the cytotoxic properties of the n-butanol extract of F. lutea with an IC50=40 μg/ml against K562 (leukemia cell line) was reported (Znati et al., 2014).
The cytotoxicity of F. assa-foetida extract on HOS CRL,an osteosarcoma cell line was also investigated. The results of this investigation showed that the cytotoxic activity of F. assa-foetida extract is dependent on the type and concentration of the solvent. Moreover, the methanol extract possessed more marked cytotoxic effects than the ethanol extract . In another study, results of MTT assay of F. assa-foetida extract against an osteosarcoma cell line (HOSCRL-1543) showed that this activity is dependent on the type of solvent (methanolic>ethanolic) and its concentration (higher methanolic content>lower methanolic content) (MohdShafri et al., 2015). Gudarzi et al. (2015) showed antiproliferative activity of ethanolic extract of F. gummosa seed, which was probably related to the presence of bioactive compounds like coumarins and terpenoids (Gudarzi et al., 2015). Additionally, cytotoxicity of hydroalcoholic extract of F. gummosa root was investigated on GP-293 cell line and primary cultured human stromal-vascular cells. The viability of human stromal-vascular cells following treatment with F. gummosa extract 400 mg/mL (60±6.5% of the control, p<0.01) and 800 mg/mL (14±1% of control, p<0.001) were significantly decreased. Also, the F. gummosa root extract reduced the viability of GP-293 cells at concentration of 750 mg/mL (8.8±0.35%, p<0.001) (Ghorbani et al., 2016).
Ferulenol, a prenylated coumarin from F. communis (Umbelliferae) exhibited tubulin-polymerizing activity. Under Ca 2+free conditions, ferulenol appeared to be equipotent as Taxol in promoting tubulin assembly (Altmann and Gertsch, 2007). Recently, it was shown that conferone 20 µM induces cell arrest and cell death through both apoptosis and necrosis in HT-29 cells (Cheraghi et al., 2016).
Galbanic acid, a sesquiterpene coumarin isolated from Ferula species showed cytotoxic activities. Galbanic acid inhibited the growth of prostate cancer cells via decreasing androgen receptor abundance (Kasaian et al., 2014). Also, galbanic acid induced apoptosis in H460 cells via caspase activation and Mcl-1 inhibition in H460 cells; therefore, it could be considered a potent cytotoxic agent against non-small cell lung carcinoma (Oh et al., 2015). Researchers also revealed that galbanic acid has anti-angiogenesis effects .
Diversin, a natural prenylated coumarin isolated from Ferula roots, revealed cytotoxic activity as well as cell-cycleinhibitory and apoptosis-inducing effects on bladder carcinoma cells (Haghighitalab et al., 2014).
Umbelliferone, a naturally occurring coumarin derivative isolated from F. communis, has been suggested as an effective cytotoxic compound against HepG2 cell line. Furthermore, umbelliferone exhibited apoptosis-inducing activity in HepG2 cells in a concentrationdependent manner (0-50 μM) (Yu et al., 2015). Huang et al. (2013) investigated two new terpenoid benzoates namely, syreiteate A and syreiteate B, isolated from the roots of F. dissecta. Their results proved that syreiteate A and syreiteate B have potent growth inhibitory activity against cervical cancer HeLa cell line with IC50 values of 13.2 and 19.3 μM, respectively (Huang et al., 2013).
Ferutinin, a natural sesquiterpene of Ferula, showed apoptosis-inducing activities in cancerous cells by induction of sub-G1 peak as revealed by PI staining . Researchers also showed that ferutinin has apoptotic effects in human Jurkat T-cell line (Macho et al., 2004).
Nano-based formulation of farnesiferol C, a sesquiterpene coumarin isolated from Ferula, significantly suppressed the proliferation of AGS gastric epithelial cells in a time-and dose-dependent manner (p<0.01). Farnesiferol C could be considered a potential chemotherapeutic agent; its anticancer effects are partly mediated via inducing tumor cells apoptosis by increasing the Bax/Bcl-2 ratio (Aas et al., 2015). Additionally, farnesiferolC isolated from the resin of F. assa-foetida L. exerted anti-angiogenic activity (Lee et al., 2010). Mousavi et al. (2015) reported auraptene apoptotic effects in MCF-7 cell line (IC50 = 59.7 µM). They revealed that auraptene induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells. In this study, DNA fragmentation was suggested as one of the underlying mechanisms of auraptene-induced apoptosis. Also, western blot analysis showed that auraptene significantly up-regulated Bax expression in MCF-7 cells compared to untreated controls (Mousavi et al., 2015).
Dietary phytochemicals present in F. assa-foetida, like luteoline, ferutinin and ferutidine, induced apoptosis and inhibited cell proliferation at the level of DNA synthesis (in S-phase) (Bansal et al., 2012;Matin et al., 2014). F. assa-foetida extract exerted anti-apoptotic activity in cerebellar granule neurons by induction of cell cycle arrest in G0/G1 phase; therefore, F. assafoetida extract was suggested to be used against neurologic disorders (Tayeboon et al., 2013). Gharaei et al. (2013) revealed that F. gummosa Boiss. extracts exerted antiproliferative as well as apoptosis-inducing effects in a human gastric adenocarcinoma cell line (AGS). They also reported that F. gummosa extracts inhibited AGS cell line proliferation in a dose-dependent manner with IC50 values of 37.47 µg/mL for flower and 32.99 µg/mL for leaf extracts. F. gummosa extracts also induced apoptosis, as reflected by DNA fragmentation and plasma membrane translocation of phosphatidyl serine (Gharaei et al., 2013). F. gummosa flower and leaf extracts inhibited angiogenesis in a concentration-dependent manner (10-30 µg/ml), reflecting the possible presence of anti-angiogenic compounds (Mirzaaghaei et al., 2014).
In another study, it was reported that F. assa-foetida and F. gummosa exert cytotoxic effects. The cytotoxic effects of F. assa-foetida were mediated through three mechanisms including inhibition of mutagenesis, DNA destruction and cancer cell proliferation, while F. gummosa exerted its effects via cell cycle arrest and induction of apoptosis (Asadi-Samani et al., 2015).
Cytotoxic activity of sesquiterpene coumarins isolated from F. nartex was examined by Alam et al. (2016). These researchers reported that n-hexane fraction of F. nartex extract shows significant cytotoxic activity against PC3 cancer cells with an IC50 value of 5.43 ± 0.24 µg/ml (Alam et al., 2016). F. vesceritensis extract, as a new natural source of lapiferin, showed promising specific cytotoxic activity against human breast cancer cells. The cytotoxic activity was shown to be mediated through induction of apoptosis. Lapiferin evoked multiple pathways involving enhancement of DNA fragmentation, activation of caspases and induction of histone acetylation, all triggering apoptosis (Gamal-Eldeen and Hegazy, 2010).
The ethyl acetate fraction of F. sinkiangensis extract revealed efficient inhibiting effects on tumor cells proliferation and enhanced the apoptosis rate in tumor cells .

Mechanisms of action
It has been found that natural agents with cell-based properties can be divided into two categories of cytotoxic and/or antiproliferative compounds (Keskin et al., 2000).
For example, sesquiterpene coumarins isolated from the Ferula genus, showed both growth inhibitory and cytotoxic activities in different cancerous cell lines (Ryuet al., 2001).
It seems that two different mechanisms of cellular growth inhibition consist of lowering proliferation rate and induction of cellular death through apoptosis or necrosis.
Generally, Bcl-2 family proteins such as Bcl-2 protein and Bax protein, have important regulatory roles in apoptosis. Aldaghi et al. indicated that farnesiferol C and microlobin, two sesquiterpene coumarins isolated from F. szowitsiana, have greater binding affinity to Bax protein in comparison to Bcl-2 protein. These researchers assumed that the interaction between drugs and hydrophobic groove of Bax protein might result in conformational changes and insertion of Bax protein into mitochondrial membrane, consequently inducing Bax-dependent apoptosis (Aldaghi et al., 2016). In another study, RT-PCR analysis of Bax and Bcl-2 genes showed that dendrosomal form of farnesiferol C could suppress AGS cell proliferation, at least in part, via inducing apoptosis. Moreover, some recent research revealed that coumarin compounds could induce apoptosis by modulating Bax/Bcl-2 and caspase pathways (Gholami et al., 2013;Sadeghizadeh et al., 2008).
Cytotoxic activity of galbanic acid was mediated through inhibiting angiogenesis, the essential process required for tumor growth and metastasis. Galbanic acid significantly decreased vascular endothelial growth factor (VEGF)-induced proliferation and inhibited VEGF-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs). These effects were accompanied by decreased phosphorylation of p38-mitogenactivated protein kinase (MAPK), c-Jun Nterminal kinase (JNK), and AKT, and decreased expression of VEGFR targets endothelial nitric oxide synthase (eNOS) and cyclin D1 in VEGF-treated HUVECs . In another study, galbanic acid showed a promising inhibitory activity against farnesyltransferase (FTase), an essential enzyme needed for tumor growth in pancreas and colon cancers (Figure 2) (Cha et al., 2011). Figure2. Overview of different mechanisms through which Ferula-isolated compounds decrease cellular growth. that much effort still remains to be made to identify potent and effective Ferula compounds that could be appropriate to be used as adjuvant therapy along with the conventional antibiotics. It is ultimately suggested that considering the versatile biological activities of sesquiterpene coumarins, these compounds may have an even broader range of biological applications in the future.